In multicellular organisms, genomic stability and integrity is maintained by the combined actions of accurate DNA replication machinery and a complex network of DNA repair pathways. DNA joining is an essential step in DNA replication, in DNA excision repair and in the repair of DNA strand breaks. Three mammalian genes encoding DNA ligases, LIG1, LIG3 and LIG4, have been identified. Genetic studies have indicated that the LIG4 gene and the functionally homologous DNL4 gene of Saccharomyces cerevisiae participate in the repair of DNA double-strand breaks (DSB)s by the non-homologous end joining (NHEJ). In mammalian cells, this pathway is required for genomic stability. Furthermore defects in NHEJ result in the type of genetic rearrangements frequently observed in cancer cells. In this proposal we will elucidate the molecular mechanisms of DNA ligase IV-dependent NHEJ in eukaryotes. Preliminary studies have revealed a functional interaction between the complex containing the yeast DNL4 gene product and its partner protein Lif 1, and the multifunctional Rad5O/Mrel l/Xrs complex. In Specific aim 1,we will employ a combination of biochemical and genetic approaches to delineate the molecular mechanisms of NHEJ in yeast. These studies will provide a conceptual framework for Specific aim 2, a parallel investigation of NHEJ in mammalian cells. In the last funding period, we identified a functional interaction between the complex containing the human LIG4 gene product and its partner protein XRCC4 and another critical NHEJ factor, the DNA-dependent protein kinase (DNA-PK). Since yeast cells lack a functional homolog of the kinase subunit of DNA-PK, it appears that there will be important differences between NHEJ in yeast and mammalian cells. The long term goal of our research on eukaryotic DNA ligases is to use these enzymes as molecular probes to gain a better understanding of the complex network of pathways that function to maintain genetic stability. The studies in this proposal will contribute to an overall picture of how the repair of DSBs by NHEJ prevents the deleterious genetic changes associated with cancer cells.